A Molecular Mechanism of Formalin Fixation and Antigen Retrieval

Sompuram, Seshi R.; Vani, Kodela; Messana, Elizabeth; Bogen, Steven A.

doi:10.1309/brn7ctx1e84nwwpl

Cited by 127 publications

(61 citation statements)

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“…22 So far, the mechanisms of crosslinking reversal as the base for antigen retrieval are not well understood. [23][24][25] Elucidation of these mechanisms will be very helpful for optimization of macromolecular extraction. Current methods for antigen retrieval include proteinase predigestion, 26 chemical pretreatment, [27][28] and heat-induced epitope retrieval.…”

Section: Discussionmentioning

confidence: 99%

A nondestructive molecule extraction method allowing morphological and molecular analyses using a single tissue section

Chu

Liang

Liu

et al. 2005

Laboratory Investigation

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In clinical practice, molecular analysis of tumor specimens is often restricted by available technology for sample preparation. Virtually all current methods require homogenization of tissues for molecule extraction. We have developed a simple, rapid, nondestructive molecule extraction (NDME) method to extract proteins and nucleic acids directly from a single fixed or frozen tissue section without destroying the tissue morphology. The NDME method is based upon exposure of micron-thick tissue section to extraction buffer with the help of heating and/or intact physical forces (ultrasound and microwave) to facilitate release of macromolecules into the buffer. The extracted proteins and nucleic acids can be used directly without further purification for downstream SDS-PAGE analysis, immunoblotting, protein array, mass spectra protein profiling, PCR, and RT-PCR reactions. Most importantly, the NDME procedure also serves as an antigen retrieval treatment, so that after NDME, the same tissue section can be used for histopathological analyses, such as H&E staining, immunohistochemistry, and in situ hybridization. Thus, the NDME method allows, for the first time, both histological diagnosis and molecular analysis on a single tissue section, whether it is from frozen or fixed tissue specimens.

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Section: Discussionmentioning

confidence: 99%

A nondestructive molecule extraction method allowing morphological and molecular analyses using a single tissue section

Chu

Liang

Liu

et al. 2005

Laboratory Investigation

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“…Ready-to-use assays offer improved reproducibility and consistency, minimizing effects arising from reagent diversity, as well as variability that can arise from antigen retrieval. [8][9][10][11][12][13][14][15][16] Surprisingly, these platform-specific ready-to-use assays have never been directly compared using the same clinical outcome series.…”

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confidence: 99%

A systematic comparison of three commercial estrogen receptor assays in a single clinical outcome breast cancer cohort

et al. 2016

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Breast cancers are routinely assessed for estrogen receptor status using immunohistochemical assays to assist in patient prognosis and clinical management. Specific assays vary between laboratories, and several antibodies have been validated and recommended for clinical use. As numerous factors can influence assay performance, many laboratories have opted for ready-to-use assays using automated stainers to improve reproducibility and consistency. Three commonly used autostainer vendors-Dako, Leica, and Ventana-all offer such estrogen receptor assays; however, they have never been directly compared. Here, we present a systematic comparison of three platform-specific estrogen receptor ready-to-use assays using a retrospective, tamoxifen-treated, breast cancer cohort from patients who were treated in Calgary, Alberta, Canada from 1985 to 2000. We found all assays showed good intra-observer agreement. Inter-observer pathological scoring showed some variability: Ventana had the strongest agreement followed closely by Dako, whereas Leica only showed substantial agreement. We also analyzed each estrogen receptor assay with respect to 5-year disease-free survival, and found that all performed similarly in univariate and multivariate models. Determination of measures of test performance found that the Leica assay had a lower negative predictive value than Dako or Ventana, compared with the original ligand-binding assay, while other measures-sensitivity, specificity, positive predictive value, and accuracywere comparable between the three ready-to-use assays. When comparing against disease-free survival, the difference in negative predictive value between the vendor assays were not as extreme, but Dako and Ventana still performed slightly better than Leica. Despite some discordance, we found that all ready-to-use assays were comparable with or superior to the ligand-binding assay, endorsing their continued use. Our analysis also allowed for exploration of estrogen receptor-negative, progesterone receptor-positive cases, and we discovered that this phenotype was not consistent across the assays, suggesting this might be an artifact. Endocrine therapy is standard treatment for hormone receptor-positive breast cancers that can improve patient survival. 1-3 Estrogen receptor and progesterone receptor analysis is routinely performed by immunohistochemical assays on breast cancer specimens to classify hormone receptor status and determine patient prognosis and management. [1][2][3][4] Eventually, immunohistochemical-based testing replaced the ligand-binding assay as the standard method for determining hormone receptor status. 5 The switch from ligand-binding assay to immunohistochemistry did not occur at a specific time point, and varied by location and laboratories between the mid-90s and early 2000s. Although estrogen and progesterone receptor are both routinely evaluated, patient management decisions are typically made on the basis of estrogen receptor results alone as the role of progesterone receptor in breast cancer patie...

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“…Sompuram and colleagues concluded that the Mannich reaction may play an important role in this process [13]. This reaction…”

Section: Discussionmentioning

confidence: 99%

Do Antigen Retrieval Techniques Improve DNA Yield from Formalin Fixed Paraffin Embedded Tissue?

Govender¹,

Naidoo²

2015

Adv Tech Biol Med

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A Molecular Mechanism of Formalin Fixation and Antigen Retrieval

Cited by 127 publications

References 0 publications

A nondestructive molecule extraction method allowing morphological and molecular analyses using a single tissue section

A nondestructive molecule extraction method allowing morphological and molecular analyses using a single tissue section

A systematic comparison of three commercial estrogen receptor assays in a single clinical outcome breast cancer cohort

Do Antigen Retrieval Techniques Improve DNA Yield from Formalin Fixed Paraffin Embedded Tissue?

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